[Histonet] Immuno staining on plastic sections

[Jack Ratliff]

A few years ago I took a workshop at the NSH presented by Neil Hand. He basically provided an account of his experience in working with something like over 100 antibodies used on resin (MMA) embedded bone and possibly other tissues. Maybe you can look his information up on the NSH website and contact him directly?

 

With regards to an "etching step", there are several posts that describe everything from HCl to formic acid to sodium ethoxide. In fact, I just found this posting from Gayle Callis back in 2004. Hope this helps!

 

Jack

 

[Histonet] Repy on immunostaining with Technovit 8100 / glycol methacrylate
Gayle Callis gcallis <@t> montana.edu 
Thu Aug 12 10:13:55 CDT 2004 

 

GMA is not a very ideal embedding media for immunohistochemistry, you would 
be far better off with paraffin using this antibody.   GMA cannot be 
removed once polymerized nor sodium ethoxide (generally reserved for 
electron microscopy resins) etched with much success, you would be better 
off embedding in methylmethacrylate and remove the plastic entirely.  This 
has been done with great success by Neil Hand (he has publications) using 
warm xylene, but he used stringent pressure cooker retrieval and worked 
with human tissue.  The problem with GMA is it prevents the immunoglobulins 
from reaching antigenic sites, as the GMA is hydrophobic plus it can't be 
removed once polymerized.  It will soften in the presence of water.

Also, as GMA polymerizes it becomes very hot due to exothermic reaction 
unless you control this temperature by letting your blocks polymerize on 
ice in a refrigerator??

GMA with IHC problems have been discussed at length on Histonet many 
times,  go to Histonet archives and search at 
www.histosearch.org.   Personally, I don't think the product "suggestion" 
is correct,  as it only "suggests" but does not say it WILL work. That is 
probably the reason you don't find it in the literature!   Many people have 
experienced failure of IHC on GMA embedded tissues. I think some have had 
success with immunofluroescence using immunoglobulins  and not a lot of 
antibodies either. It was a tedious stringent protocol described in a 
symposium talk.     I think you will find in Histonet commentary, that most 
people attempting IHC/GMA suggest going to another embedding media.

Also, CD68 ED1 is a Mouse antiRat, rather than a rat antiMouse (clone 
FA-11).  ED1 and other rat macrophage markers, ED2, ED3, have been 
discussed on Histonet as FFPE tissues including retrieval for IHC.  The 
staining was very straightforward as was retrieval described and solvents 
used plus heat of paraffin processing did not damage antigen.

ED1, when used in on rat tissue, works well on paraffin sections, although 
we prefer frozen sections to avoid all aldehyde fixation and no 
retrieval.  When we do frozen sections, the ED1 is diluted out 1:3000 or 
so, it will not be so dilute for paraffin sections and we detected with 
secondary to mouse IgG1 isotype (Southern Biotechnology)  SA-HRP, and AEC 
chromogen.  Staining pattern is spectacular in a rat spleen, normal 
positive control.

Gayle Callis
MT,HT,HTL(ASCP)
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610


 
> To: histonet <@t> lists.utsouthwestern.edu
> Date: Fri, 9 Apr 2010 09:11:59 -0400
> From: bruski33 <@t> aol.com
> Subject: [Histonet] Immuno staining on plastic sections
> 
> 
> 
> We are trying to do immuno staining on plastic sections and are not having much luck. Does anyone have any experience with this? We are embedding in technovit 8100. It is a device with encapsulated VEGFR cells. I have been told that we might have to do an etching step. Any help would be greatly appreciated.
> Bruce
> 
> 
> 
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